Limits...
Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9.

Picconi JL, Muff-Luett MA, Wu D, Bunchman E, Schaefer F, Brophy PD - Mol Ther Methods Clin Dev (2014)

Bottom Line: Gene therapy targeting of kidneys has been largely unsuccessful.An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life.Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics , Iowa City, Iowa, USA ; Department of Pediatrics, University of Iowa Children's Hospital , Iowa City, Iowa, USA.

ABSTRACT
Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV) vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP) gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence staining for GFP in kidneys of treated pups at 8 weeks. Pregnant adult dams at E12.5 were treated with 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1), saline control (n = 1), or 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) by tail vein injection. Pups (a: rAAV2/9; n = 12), (b: rAAV2; n = 9), and saline n = 10 (not shown) were sacrificed at 8 weeks of life. Dams (c: rAAV2; d: rAAV2/9, and e: saline control) were sacrificed at 8 weeks postinjection. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunofluorescence staining for DAPI (blue), GFP (green), WT-1 (red), and merge slide. Bars = 50 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362350&req=5

fig3: Immunofluorescence staining for GFP in kidneys of treated pups at 8 weeks. Pregnant adult dams at E12.5 were treated with 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1), saline control (n = 1), or 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) by tail vein injection. Pups (a: rAAV2/9; n = 12), (b: rAAV2; n = 9), and saline n = 10 (not shown) were sacrificed at 8 weeks of life. Dams (c: rAAV2; d: rAAV2/9, and e: saline control) were sacrificed at 8 weeks postinjection. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunofluorescence staining for DAPI (blue), GFP (green), WT-1 (red), and merge slide. Bars = 50 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.

Mentions: When kidneys from the rAAV 2/9-CMV-eGFP–treated pups were examined, robust staining was identified (Figure 2a, IH and Figure 3a, IF). Specifically, glomeruli and tubules were clearly shown to be expressing GFP. In contrast, the kidneys of pups from rAAV2-CMV-eGFP–treated animals demonstrated no identifiable staining (Figure 2b, IH and Figure 3b, IF). Kidneys taken from the treated dams showed the same patterns of IF and IH staining as the pups, with absence of staining in kidneys of the dams treated with rAAV2-CMV-eGFP (Figure 2c, IH and Figure 3c, IF), similar to saline-injected controls (Figure 2e, IH and Figure 3e, IF). Robust staining was also seen in the kidneys of the dams treated with rAAV 2/9-CMV-eGFP (Figure 2d, IH and Figure 3d, IF).


Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9.

Picconi JL, Muff-Luett MA, Wu D, Bunchman E, Schaefer F, Brophy PD - Mol Ther Methods Clin Dev (2014)

Immunofluorescence staining for GFP in kidneys of treated pups at 8 weeks. Pregnant adult dams at E12.5 were treated with 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1), saline control (n = 1), or 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) by tail vein injection. Pups (a: rAAV2/9; n = 12), (b: rAAV2; n = 9), and saline n = 10 (not shown) were sacrificed at 8 weeks of life. Dams (c: rAAV2; d: rAAV2/9, and e: saline control) were sacrificed at 8 weeks postinjection. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunofluorescence staining for DAPI (blue), GFP (green), WT-1 (red), and merge slide. Bars = 50 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362350&req=5

fig3: Immunofluorescence staining for GFP in kidneys of treated pups at 8 weeks. Pregnant adult dams at E12.5 were treated with 8.4 × 1012 viral genomes rAAV 2/9-CMV-eGFP (n = 1), saline control (n = 1), or 1.3 × 1012 viral genomes rAAV2-CMV-eGFP (n = 1) by tail vein injection. Pups (a: rAAV2/9; n = 12), (b: rAAV2; n = 9), and saline n = 10 (not shown) were sacrificed at 8 weeks of life. Dams (c: rAAV2; d: rAAV2/9, and e: saline control) were sacrificed at 8 weeks postinjection. Kidneys were snap frozen, whole mounted for imaging, cryosectioned (10 μm), and processed for immunofluorescence staining for DAPI (blue), GFP (green), WT-1 (red), and merge slide. Bars = 50 μm. CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein.
Mentions: When kidneys from the rAAV 2/9-CMV-eGFP–treated pups were examined, robust staining was identified (Figure 2a, IH and Figure 3a, IF). Specifically, glomeruli and tubules were clearly shown to be expressing GFP. In contrast, the kidneys of pups from rAAV2-CMV-eGFP–treated animals demonstrated no identifiable staining (Figure 2b, IH and Figure 3b, IF). Kidneys taken from the treated dams showed the same patterns of IF and IH staining as the pups, with absence of staining in kidneys of the dams treated with rAAV2-CMV-eGFP (Figure 2c, IH and Figure 3c, IF), similar to saline-injected controls (Figure 2e, IH and Figure 3e, IF). Robust staining was also seen in the kidneys of the dams treated with rAAV 2/9-CMV-eGFP (Figure 2d, IH and Figure 3d, IF).

Bottom Line: Gene therapy targeting of kidneys has been largely unsuccessful.An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life.Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics , Iowa City, Iowa, USA ; Department of Pediatrics, University of Iowa Children's Hospital , Iowa City, Iowa, USA.

ABSTRACT
Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV) vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP) gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV)-enhanced green fluorescent protein (eGFP) vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

No MeSH data available.


Related in: MedlinePlus